1. Field of the Invention
The invention relates generally to the field of diagnostic assays. More particularly, the present invention relates to the use of an analyte-substitute reagent in a binding assay to form a detectable binding member complex, wherein that detectable complex correlates to the presence or amount of analyte in the test sample.
2. Description of Related Art
Various analytical procedures and devices are commonly employed in assays to determine the presence and/or concentration of substances of interest or clinical significance which may be present in biological fluids or other materials. Such substances are commonly termed "analytes" and can include antibodies, antigens and haptens. In the diagnosis and treatment of disease or other conditions of the human body, the accurate and timely determination of the presence or amount of an analyte in a biological sample can have a profound influence on the ability of health care professionals to treat and manage a pathological disorder. In addition, the performance of such assays enables early and accurate determination of physiological conditions such as pregnancy, the monitoring of drug therapy and the evaluation of the absence, presence and amount of drugs of abuse or toxins.
Typical immunoassay techniques utilize the mechanisms of the immune systems of higher organisms, wherein antibodies are produced in response to the presence of antigens which are pathogenic or foreign to the organisms. One or more antibodies are produced in response to and are capable of reacting with a particular antigen, thereby creating a highly specific reaction mechanism which can be used in vitro to determine the presence or concentration of that particular antigen in a biological sample.
Heterogeneous immunoassay techniques typically involve the use of a solid phase material to which the reaction product becomes bound. The reaction product is separated from excess sample, assay reagents and other substances by removing the solid phase from the reaction mixture. One type of solid phase immunoassay is a sandwich immunoassay involving an anti-analyte antibody (capture reagent) bound to the insoluble solid phase material, as described by Schuurs et al. U.S. Pat. Nos. 3,791,932 and 4,016,043. A second anti-analyte antibody is labeled with a detectable agent to form an indicator reagent, e.g., the detectable label can be an enzyme which will react with an enzyme substrate to form a detectable product. If the analyte is present in the test sample, then the two antibodies form an immunocomplex with the analyte (i.e., an antibody/analyte/antibody sandwich), and the amount of indicator reagent associated with the solid phase is directly proportional to the amount of analyte in the test sample. When the enzyme substrate is added, it reacts with the enzyme component of the indicator reagent to signal the presence or amount of analyte associated with the solid phase.
Another type of solid phase immunoassay configuration is the competitive assay. In a manner similar to the sandwich assay, the competitive assay can involve an anti-analyte antibody bound to the insoluble solid phase, but a labeled analyte, instead of a labeled second antibody, may be used as the indicator reagent. In the competitive assay, the indicator reagent competes with the test sample analyte to bind the capture reagent on the solid phase. The amount of captured indicator reagent is inversely proportional to the amount of analyte present in the test sample. Smith (U.S. Pat. No. 4,401,764) describes an alternative competitive assay format using a mixed binding complex which can bind analyte or labeled analyte but wherein the analyte and labeled analyte can not simultaneously bind the complex. Clagett (U.S. Pat. No. 4,746,631) describes an immunoassay method using a reaction chamber in which an analyte/ligand/marker conjugate is displaced from the reaction surface in the presence of test sample analyte and in which the displaced analyte/ligand/marker conjugate is immobilized at a second reaction site. The conjugate includes biotin, bovine serum albumin and synthetic peptides as the ligand component of the conjugate, and enzymes, chemiluminescent materials, enzyme inhibitors and radionucleotides as the marker component of the conjugate. Li (U.S. Pat. No. 4,661,444) describes a competitive immunoassay using a conjugate of an anti-idiotype antibody and a second antibody, specific for a detectable label, wherein the detectable response is inversely related to the presence of analyte in the sample.
In both the sandwich and competitive immunoassays, the presence or amount of analyte in the test sample is generally determined by detecting the presence or amount of the label which has become associated with the solid phase. In the competitive assay, the more analyte present in the test sample the lower the amount of label present on the solid phase. In the sandwich assay, the more analyte present in the sample the greater the amount of label present on the solid phase. The sandwich assay is generally preferred, especially for the visualization of low analyte concentrations, because the appearance of label on the solid phase is more readily detected.
The sandwich assay, however, is subject to several limiting factors. Certain analytes of interest, such as some steroids, hormones, antibiotics and other therapeutic drugs, have a low molecular weight and a corresponding size that is too small to allow the simultaneous binding of two antibodies to the analyte and thereby form the sandwich complex. Methods for detecting such analytes typically use a competitive assay configuration wherein the analyte either competes with an indicator reagent for binding to a capture reagent or wherein the analyte displaces the indicator reagent from the capture reagent. A positive result in a displacement assay is indicated by a decrease in the amount of label associated with the solid phase material. Visually determining a positive assay result by detecting a decreasing amount of label is more difficult than detecting the appearance of label on the solid phase, and difficulty in discerning a decrease in the amount of label can lead to ambiguity in the interpretation of the assay result. Allen (EP 177,191) describes a binding assay involving a conjugate of a ligand analog and a second reagent, such as fluorescein, wherein the conjugate competes with the analyte (ligand) in binding to a labeled binding partner specific for the ligand, and wherein the resultant labeled conjugate is then separated from the reaction mixture by means of solid phase carrying a binding partner for the second reagent. This binding assay format combines the use of a competitive binding technique and a reverse sandwich assay configuration, i.e., the binding of conjugate to the labeled binding member prior to separating conjugate from the mixture by the binding of the conjugate to the solid phase. The assay result, however, is determined as in a conventional competitive assay wherein the amount of label bound to the solid phase is inversely proportional to the amount of analyte in the test sample. Chieregatt et al. (GB 2,084,317) describe a similar assay format using an indirectly labeled binding partner specific for the analyte. Mochida et al. (U.S. Pat. No. 4,185,084) also describe the use of a double-antigen conjugate which competes with an antigen analyte for binding to an immobilized antibody and which is then labeled; this method also results in the detection of label on a solid phase wherein the amount of label is inversely proportional to the amount of analyte in the test sample. Sadeh et al. (U.S. Pat. No. 4,243,749) describe a similar enzyme immunoassay wherein a hapten conjugate competes with analyte for binding to an antibody immobilized upon a solid phase.